X chromosome inactivation (XIC) is the pre-eminent model for formation of facultative heterochromatin in mammalian cells, a process central to genome regulation in early embryos, and relevant to its abrogation in cancer. In pluripotent stem cells, a remarkable, large non-coding RNA from the X-linked XIST gene accumulates and spreads across one female X chromosome, thereby initiating a process which transforms the euchromatic chromosome into a dense, heterochromatic Barr Body. While much has been learned about histone modifications which mark heterochromatin, it is still not understood what specifically silences transcription. Further, it remains a mystery how XIST RNA localizes to and paints the structure of its parent chromosome, and how this leads to the cascade of biochemical, higher-order structural, and transcriptional changes of a whole chromosome. The fundamental principles are directly relevant to all chromosomes, since autosomes can be silenced by XIST. Although studies of the silencing process have been largely restricted to mouse ES/transgenic cells, we recently demonstrated successful creation of the first tractable, robust model that fully emulates human chromosome silencing. This advantageous new system, in the pluripotent cell context, provides a strong foundation for this project, and a needed resource to the field. We will examine novel concepts that inter-relate non-coding RNA, chromosome architecture, and genomic sequence, at progressive steps during the chromosome remodeling cascade. Our studies have the potential to change the existing view that interspersed repetitive DNA, comprising almost half the human genome, is unimportant for genome function. Our recently published findings demonstrate a new class of chromosomal RNA, rich in repeat sequences and associated with euchromatin. They further suggest that abundant, widely distributed chromosomal RNAs in active chromosomes serve to promote open chromatin state. The research strategy integrates a range of cutting-edge approaches, from in situ analysis of RNAs or DNAs within nuclear structure, to sophisticated gene targeting, high through-put screening, in vitro RNA biochemistry, and bioinformatics. Aim 1 is focused on dissecting the inter-relationships between key events in the chromosome silencing process, in a new system to initiate human chromosome silencing in patient iPS cells Aim 2 will define the chromosomal anchor proteins and domains of human XIST RNA required for its localization and function, and test a novel hypothesis regarding competition between chromosomal RNAs. Aim3 investigates the role of chromosome sequence context, particularly different repeat families, in XIST RNA function, and its escape. Research proposed has high potential at multiple levels to advance understanding of the fundamental structure of chromosomes, the DNA sequences involved, and the RNAs/proteins integral to their higher-order structure.